Our long-term goal is to understand GABAergic inhibition in neurological disorders. GABAA receptors (GABAA-Rs) mediate the majority inhibition in the brain. Synaptically localized GABAA-Rs mediate fast inhibitory neurotransmission. Extrasynaptic GABAA-Rs can be activated by a low concentration of ambient GABA to mediate tonic inhibition, which plays an important role in regulating neuronal excitability. Extrasynaptic GABAA-Rs play important roles in learning and memory, anxiety and depression, alcoholism, epilepsy, and sleep disorders. Despite our increasing understanding of the functional significance of extrasynaptic GABAA-Rs, the molecular mechanisms underlying extrasynaptic targeting of GABAA-Rs are poorly understood. The specific objective of this proposal is to unravel the targeting mechanisms of d subunit-containing GABAARs, which is the major subtype of extrasynaptically localized GABAARs. Based on a strong set of preliminary data, we propose a novel hypothesis that in addition to ?2 and d subunits, a subunits also carry critical targeting signals governing synaptic versus extrasynaptic localization of GABAARs. This hypothesis will be tested with a unique model system of molecularly reconstituted functional GABAergic synapses. With precise control of the postsynaptic compositions in model synapses, we will investigate how synaptic and extrasynaptic GABAARs interact with neuroligin-2/gephyrin/collybistin complex. Immuno-electron microscopy and quantum dot-based time lapse imaging will be used to visualize the localization and dynamic movement of d-GABAARs. Finally, ?2-/- and d-/- neurons will be used to investigate functional rescue of chimeric and mutant d-GABAARs and ?2-GABAARs. Our studies will gain fundamental understanding of the extrasynaptic targeting mechanisms of d-GABAA-Rs, which is pivotal for molecular designing of new drug therapy to treat neurological disorders that are associated with extrasynaptic GABAA-Rs.